Involvement of β‐adrenoceptors in the cardiovascular responses induced by selective adenosine A2A and A2B receptor agonists

Abstract A2A and A2B adenosine receptors produce regionally selective regulation of vascular tone and elicit differing effects on mean arterial pressure (MAP), whilst inducing tachycardia. The tachycardia induced by the stimulation of A2A or A2B receptors has been suggested to be mediated by a reflex increase in sympathetic activity. Here, we have investigated the role of β1‐ and β2‐adrenoceptors in mediating the different cardiovascular responses to selective A2A and A2B receptor stimulation. Hemodynamic variables were measured in conscious male Sprague‐Dawley rats (350–450 g) via pulsed Doppler flowmetry. The effect of intravenous infusion (3 min per dose) of the A2A‐selective agonist CGS 21680 (0.1, 0.3, 1.0 µg.kg−1.min−1) or the A2B‐selective agonist BAY 60–6583 (4.0, 13.3, 40.0 µg.kg−1.min−1) in the absence or following pre‐treatment with the non‐selective β‐antagonist propranolol (1.0 mg.kg−1), the selective β1‐antagonist CGP 20712A (200 µg.kg−1), or the selective β2‐antagonist ICI 118,551 (2.0 mg.kg−1) was investigated (maintenance doses also administered). CGP 20712A and propranolol significantly reduced the tachycardic response to CGS 21680, with no change in the effect on MAP. ICI 118,551 increased BAY 60–6583‐mediated renal and mesenteric flows, but did not affect the heart rate response. CGP 20712A attenuated the BAY 60–6583‐induced tachycardia. These data imply a direct stimulation of the sympathetic activity via cardiac β1‐adrenoceptors as a mechanism for the A2A‐ and A2B‐induced tachycardia. However, the regionally selective effects of A2B agonists on vascular conductance were independent of sympathetic activity and may be exploitable for the treatment of acute kidney injury and mesenteric ischemia.


| INTRODUC TI ON
Adenosine is a purine nucleoside that has an important role within the cardiovascular system. 1,2 The physiological actions of adenosine are the consequence of its interaction with four different G proteincoupled receptors (GPCRs), namely the adenosine A 1 , A 2A , A 2B , and A 3 receptors (A 1 R, A 2A R, A 2B R, A 3 R). 3,4 By interacting with these GPCRs, adenosine and its analogues initiate defined signaling pathways that provoke different biological effects on numerous organ systems. 5,6 A 1 and A 3 adenosine receptors primarily couple to the inhibitory G-proteins G o or G i suppressing cyclic adenosine monophosphate (cAMP) production, while, in contrast, A 2A and A 2B subtypes preferentially activate stimulatory G s proteins, thus increasing intracellular cAMP concentrations. 4,7 A 2A and A 2B receptors are widely expressed in the cardiovascular system, where their regulation plays a key modulatory role in controlling heart rate and blood pressure. [8][9][10] In addition to regulating heart rate (HR), cardiac contraction, inflammation, and vascular remodeling, 3,11 both A 2A and A 2B receptors mediate systemic and pulmonary vasodilation. 12,13 Activation of these adenosine receptors in response to hypoxic or ischemic stress also plays an important role in the prevention of renal failure by promoting renal perfusion. 7,14 A 2A and A 2B receptors are therefore promising targets for a wide range of cardiovascular diseases, in particular hypertension and acute kidney injury. 7,14,15 An in vivo evaluation of the cardiovascular effects of selective A 2A and A 2B agonists, CGS 21680 and BAY 60-6583, respectively, showed that A 2A and A 2B receptors exert a regionally selective control of vascular conductance. 10 The A 2A R subtype mediates vasodilatory effects in the hindquarters vascular bed, with minimal impact on the mesenteric and renal vasculature, whereas A 2B Rs have been demonstrated to have major control of renal and mesenteric vascular tone but have no effect on hindquarters vascular conductance. 10 In addition, the activation of both A 2 receptor subtypes resulted in a parallel increase in HR, which may be secondary to a reflex response to the vasodilatation induced in different vascular beds. 10 The arterial baroreflex is a neural mechanism that plays a crucial role in the fine regulation of blood pressure. 16 The continuous sensation of blood pressure by tonic arterial baroreceptors allows this reflex to make constant adjustments of blood pressure by inducing rapid changes in heart rate and peripheral vascular resistance. 17 Within the context of the neural control of cardiovascular function, the nucleus of the solitary tract (NTS) represents the first synaptic station for the processing of cardiovascular afferent inputs. 18 In this regard, there is evidence of A 2A receptor involvement in the control of baroreflex activity via NTS, 19 as well as A 2B receptor involvement in cardiovascular regulation via the posterior hypothalamus. 20 Sympathetic activity and its effect on the cardiovascular system are crucial aspects of the baroreceptor reflex. 16 Indeed, the modulation of the sympathetic outflow is under the control of baroreceptor afferent activity, which evokes changes in the sympathetic activity to maintain an adequate blood pressure. 21 The physiological responses to sympathetic activation result from the interaction between catecholamines and adrenoceptors. 22 In particular, β 1 and β 2 adrenoceptors play fundamental roles in the regulation of cardiovascular homeostasis. 23,24 To evaluate the contribution of β adrenoceptors to the tachycardia induced by selective A 2A

| Animals and surgery
Male Sprague-Dawley rats (Charles River Laboratories, UK; 350-450 g) were used to perform these experiments. Animals were housed in pairs in a temperature-controlled room (21-23°C) with a 12 h light-dark cycle (lights on at 06:00) with free access to food (18% Protein Rodent Diet; Envigo, Madison WI, USA) and water. Upon arrival within the Unit, animals were housed during an acclimatization period of at least of 7 days prior to any surgery. All procedures were performed with approval from the University of Nottingham Animal Welfare and Ethical Review Board and performed in line with the Animals (Scientific Procedures) Act (1986), under UK Home Office approved Project License and Personal License authority. 53 rats were used during this study, and all animal experiments are reported in compliance with the ARRIVE guidelines 25 and the editorial on reporting animal studies. 26 Surgical procedures were carried out under general anesthesia (fentanyl and medetomidine, 300 µg.kg −1 each, i.p., supplemented as required). During the first surgery, miniature pulsed Doppler flow probes were implanted around the left renal and superior mesenteric arteries and the descending abdominal aorta to monitor haemodynamics. 27 The probe wires were led subcutaneously to the nape of the neck, where they were taped and secured. Atipamezole hydrochloride (1 mg.kg −1 , s.c.) and buprenorphine (30 µg.kg −1 , s.c.) were provided as reversal agents and postoperative analgesia. A second dose of analgesia (buprenorphine 15 µg.kg −1 , s.c.) was given 4 h post-surgery.
A second surgery was carried out at least 10 days after the surgical implantation of the vascular probes and after a satisfactory welfare inspection from the Named Veterinary Surgeon. During this surgery, performed under anesthesia (fentanyl and medetomidine, 300 µg.kg −1 each, i.p., supplemented as required), a catheter was implanted into the distal abdominal aorta via the caudal artery (to measure arterial blood pressure and heart rate), and three catheters were implanted into the right jugular vein (for drug administration). 27 All catheters were led subcutaneously to the nape of the neck.
The probe wires were released from the nape of the neck to be soldered into a miniature plug (Omnetic connector corporation, USA), which was then mounted onto a custom-designed harness worn by the rat. The catheters and probe wires were protected by a spring secured to the harness and attached to a counterbalanced pivot system to allow the free movement of the animal. Reversal of anesthetic and analgesia was administered (as described above). The arterial catheter was filled and infused with heparinized (15 U.ml −1 ) saline overnight to maintain potency.
Experiments began 24 h after surgery for catheter implantation, with animals fully conscious and unrestrained in home cages, with free access to food and water.

| Cardiovascular recordings
During the cardiovascular monitoring periods, rats were connected to the customized data-acquisition software (see below) via a tether

| Experimental protocol
Experiments were run in six studies, each lasting 3 days; within each study was a contemporaneous vehicle control (5% propylene glycol, 2% Tween 80 in sterile saline). Experiments were run with treatment groups of 8 to 10 rats.   Ten animals were used to assess the cardiovascular responses to BAY 60-6583 in the presence or absence of propranolol. After a period of baseline recordings, rats were randomized into two groups.
Group 1 received vehicle intravenous bolus (0.1 ml provided over Hemodynamic recordings were made for a further 4 h following the completion of the BAY 60-6583 intravenous infusion period.

| NanoBRET ligand binding studies
The evaluation of the binding of β-adrenoceptor ligands to rat

| Data analysis
All in vivo data were collected and analysed using IdeeQ software

| Effect of β-adrenoceptor antagonists on the hemodynamic profile of the A 2A agonist CGS 21680
Consistent with our previous observations, 10 Figure 2). However, no significant difference was observed in MAP,

RVC, MVC, and HVC between vehicle-pretreated rats and CGP
20712A-pretreated rats ( Figure 2). This dose of CGP 20712A was selected as it has previously been shown to produce a highly selective antagonism of cardiac β 1 -adrenoceptors in vivo in the rat. 30,31 Selective antagonism of β 2 -adrenoceptors with ICI 118,551, 30,31 however, produced a small but significant increase in the RVC response mediated by CGS 21680, but did not alter HVC, HR, or MAP ( Figure 3). Pretreatment with the non-selective β-blocker propranolol produced a marked (p < .05) decrease in HR with no significant change in MAP (Figure 4). Propranolol did, however, produce a small reduction (p < .05) in the hindquarters vasodilatation ( Figure 4).

| Effect ofβ-adrenoceptor antagonists on the hemodynamic profile of the A 2B agonist BAY 60-6583
As noted previously, 10

| NanoBRET ligand binding studies
To verify that the β-adrenoceptor ligands do not directly bind to either of the two A 2 adenosine receptors, we also performed NanoBRET ligand binding studies utilizing rat N-terminal Nanoluciferase-tagged A 2A and A 2B receptors, as described previously. 10  100 μM, suggesting that none of the β-adrenoceptor ligands tested directly bind to either of the two rat A 2 receptors.

| DISCUSS ION
Consistent with our previous observations, 10   has been shown to improve cardiac function by preventing the remodeling and fibrogenesis that occurs following A 2B receptor activation. 52 Interestingly, tissue-specific knockout of A 2B receptors from both cardiomyocytes and vascular endothelial cells showed that A 2Breceptors were critical for ischemia-reperfusion injury-elicited cardioprotection, but reperfusion injury was also increased if A 2B -receptor signaling was knocked out from inflammatory cells. 53 Selective activation of A 2B -receptors has also been demonstrated to play an import- tection are yet to be fully defined. As a result, addressing the specific role of A 2B signaling in renal ischemia-reperfusion injury might be crucial to define the potential therapeutic uses of A 2B receptor agonists in the context of renal injury.
In summary, the present study has confirmed that the tachycardia induced by the selective A 2A -receptor agonist CGS 21680 is partly due to an activation of the sympathetic nervous system, and can be readily attenuated by the selective β 1 -adrenoceptor antagonist CGP 20712A.

| Study limitations
Although in this rodent model our findings indicate that selective investigated. All four adenosine receptor subtypes are known to undergo agonist-induced desensitization, internalization, and cellular trafficking, the effect of which may not be seen in these short-term in vivo studies. 59,60 Therefore, future studies should include longer term dosing regimens and observations, in addition to clinical studies, to confirm that the conclusions presented here translate to the human condition.

D I SCLOS U R E S
The authors declare no conflicts of interest.

E TH I C S S TATEM ENT
All procedures were performed with approval of the University

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.